A multitude of methods is known in the art for processing biological tissue for microscopic examination, often collectively referred to as “histotechnology.” Such methods are in worldwide use nowadays and have become of paramount importance for countless applications of microscopy in basic science, clinical science, clinical routine, toxicology, and pharmaceutical and biotechnological research and development. For instance, staining tissue specimens/sections with dyes and/or processing them with immunohistochemistry (IHC) is used in virtually every clinical pathology laboratory for the diagnosis of abnormal cells such as those found in cancerous tumors. IHC is also used in almost every biomedical research laboratory to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue. For instance, specific molecular markers are characteristic of particular cell types, such as CD8 for cytotoxic T cells and NeuN for neurons (to mention only a few). Other molecular markers are characteristic of particular cellular events, such as proliferation (addressing the cell cycle) or cell death (apoptosis). RNA in situ hybridization (ISH) can be used to localize and measure messenger RNAs (mRNAs) and other DNA transcripts within tissue sections or whole mounts of tissue specimens. DNA ISH can be used to determine the structure of chromosomes. A common application of fluorescent DNA ISH (FISH) is the assessment of chromosomal integrity in medical diagnostics. As those skilled in the art will appreciate, the present description of applications of the methods for processing biological tissue for microscopic examination is intended in an illustrative rather than in a limiting sense.
Despite their worldwide use in countless applications of microscopic analysis, none of the conventional methods known in the art for processing biological tissue for microscopic examination can be considered perfect. For instance, washing tissue specimens/sections can result in incomplete and/or uneven cleaning; fixing tissue specimens/sections can result in incomplete and/or uneven fixation and/or even or uneven over-fixation; dehydrating tissue specimens/sections can result in incomplete and/or uneven dehydration and/or even or uneven over-dehydration; hydrating tissue specimens/sections can result in incomplete and/or uneven hydration and/or even or uneven over-hydration; clearing tissue specimens/sections can result in incomplete and/or uneven clearing and/or even or uneven over-clearing; embedding tissue specimens/sections into an embedding medium can result in incomplete and/or uneven embedding and/or even or uneven over-embedding; mounting tissue specimens/sections with a mounting medium can result in incomplete and/or uneven mounting and/or even or uneven overmounting; cryoprotecting tissue specimens/sections can result in incomplete and/or uneven cryoprotection and/or even or uneven over-cryoprotection; freezing tissue specimens/sections can result in incomplete and/or uneven freezing and/or even or uneven over-freezing; thawing tissue specimens/sections can result in incomplete and/or uneven thawing and/or even or uneven over-heating; removing embedding medium from tissue specimens/sections can result in incomplete and/or uneven removing of the embedding medium and/or even or uneven removing of more than just the embedding medium; staining tissue specimens/sections can result in incomplete and/or uneven staining and/or even or uneven over-staining; processing tissue specimens/sections with histochemistry can result in incomplete and/or uneven histochemical processing and/or even or uneven over-processing; processing tissue specimens/sections with immunohistochemistry (IHC) and/or fluorescence immunohistochemistry/immunofluorescence (IF) can result in incomplete and/or uneven detection of antigens and/or even or uneven cross-reactions with other antigens to which the applied antibodies and/or antibody mimetics do not bind specifically, as well as in incomplete and/or uneven counterstaining and/or even or uneven over-counterstaining in IHC and IF, and processing tissue specimens/sections with in situ hybridization (ISH) and/or fluorescence in situ hybridization (FISH) can result in incomplete and/or uneven hybridization of labeled complementary DNA or RNA strands to specific DNA or RNA sequences and/or even or uneven cross-reactions with other DNA or RNA sequences to which the applied labeled complementary DNA or RNA strands do not hybridize specifically, as well as in incomplete and/or uneven counterstaining and/or even or uneven over-counterstaining in ISH and FISH.
Moreover, the various sub-steps of washing, fixing, dehydrating, hydrating, clearing, embedding, mounting, cryoprotecting, freezing, thawing, and/or staining tissue specimens/sections, removing embedding medium from tissue specimens/sections, and/or processing tissue specimens/sections with histochemistry, IHC, IF, ISH and/or FISH can last between a few seconds and several months and are, thus, very time-consuming as well as tedious and cumbersome. Besides this, the various sub-steps require the use of various chemicals, media, antibodies, antibody mimetics, and labeled DNA or RNA strands, many of which cost between a few dollars and thousands of dollars and, thus, are very expensive. Additionally, many of the chemicals used in the various sub-steps are regarded as being toxic and, thus, pose a potential threat not only to the health and life of those performing the methods for processing biological tissue for microscopic examination, but also to the environment. As those skilled in the art will appreciate, the present description of shortcomings, time requirements, costs, and hazard potential of the conventional methods for processing biological tissue for microscopic examination is intended in an illustrative rather than in a limiting sense.
Several methods are known that aim at improving individual histotechnological procedures for these various sub-steps for microscopic examination. For instance, U.S. Pat. No. 5,244,787 to Key et al., issued on Sep. 14, 1993, teaches a method of immunologically staining a formalin-fixed tissue specimen that comprises subjecting a formalin-fixed tissue specimen to microwave energy while the tissue specimen is submersed in water for a time sufficient to increase immunostaining efficiency (known in the art as “antigen retrieval”), removing the tissue specimen from the water and cooling, and contacting the tissue specimen with an immunological staining reagent. U.S. Pat. No. 5,578,452 to Shi et al., issued on Nov. 26, 1996, teaches a method for restoring immunoreactivity of a tissue specimen fixed with an aldehyde fixing agent and embedded in an embedding medium by contacting the tissue specimen with a solvent for the embedding medium and an aldehyde releasing reagent, which reagent releases aldehyde from the tissue by reacting the aldehyde in a substantially irreversible manner to form a non-aldehyde derivative, and removing or neutralizing excess aldehyde releasing reagent from the tissue specimen. U.S. Pat. No. 7,067,325 to Christensen et al., issued on Jun. 27, 2006, teaches an automated method of removing paraffin based embedding medium from tissue specimens without the dependence on organic solvents by applying a deparaffinizing and antigen retrieval reagent that includes a detergent to the tissue specimen, and applying heat to the tissue specimen to melt the paraffin based embedding medium, to mention only a few.
However, none of these methods have addressed known histotechnology procedures, such as the sub-steps listed above, as a whole, and the application of certain chemicals and particularly the application of heat during tissue processing can challenge tissue integrity substantially and, thus, clarity and detectability of microscopic details when examining tissue specimens/sections under a microscope.
Recently, a number of ultrasound-based methods were described that aim at improving one or more of the known histotechnology procedures. The term “ultrasound” usually refers in the art to cyclic sound pressure/acoustic waves having a frequency greater than the upper limit of human hearing, i.e., typically above 20 KHz. For example, U.S. Pat. No. 3,961,097 to Gravlee, issued on Jun. 1, 1976, teaches a method for preparing tissue specimens for microscopic examination, including the steps of fixing, dehydrating, clearing, and impregnating tissue specimens with paraffin, and applying low frequency ultrasound (50 KHz) to tissue specimens in each of these processing steps in order to reduce the total preparation time. Likewise, U.S. Pat. No. 5,089,288 to Berger, issued on Feb. 18, 1992, teaches a method for impregnating tissue specimens with paraffin, including the steps of fixing, dehydrating, and embedding tissue specimens in paraffin, with exposure of the tissue specimen to low frequency ultrasound (35-50 KHz) during paraffin embedding in a closed, evacuated working chamber. However, these methods suffer from potential tissue damage due to the low frequency of the applied ultrasound, as demonstrated in many studies reviewed in U.S. Pat. No. 7,090,974 to Chu, issued on Aug. 15, 2006 (“the '974 patent”).
The '974 patent is part of a series of related U.S. patents issued to Chu, all entitled “Ultrasound-mediated high-speed biological reaction and tissue processing” (U.S. Pat. No. 6,291,180 issued on Sep. 18, 2001; U.S. Pat. No. 7,090,974 issued on Aug. 15, 2006; U.S. Pat. No. 7,262,022 issued on Aug. 28, 2007; U.S. Pat. No. 7,687,255 issued on Mar. 30, 2010; and U.S. Pat. No. 7,767,434 issued on Aug. 3, 2010), and teaches a method that is directed to using high intensity, high frequency, nondestructive, wide-band ultrasound for tissue fixation and processing in conjunction with well-known techniques to decrease the time required to perform the techniques, including immunological reactions, hybridizations, tissue fixation and processing. According to the Chu method, the tissue specimens/sections must receive at least 10 W/cm2 of fairly even distributed ultrasound using a single high frequency or using wide-band frequencies within the range of 0.1-50 MHz.
However, the Chu method suffers from several drawbacks. First, it requires very special and presumably very expensive technology to produce ultrasound transducers that are capable of delivering such high-frequency, high-intensity ultrasound. Second, the '974 patent discloses that the high-frequency, high-intensity ultrasound exposes tissue specimens/sections to heat and, thus, can challenge tissue integrity substantially and, thus, clarity and detectability of microscopic details when examining tissue specimens/sections under a microscope. Third, the Chu method does not disclose any improvement over prior art with respect to reducing the amount of media and/or chemicals necessary to carry out one or more or all steps of the procedures of histotechnology (except of dyes, antibodies and/or antibody mimetics, and/or labeled DNA and/or RNA strands), with many of these media and chemicals regarded being toxic. Fourth, the method does not disclose any improvement over prior art with respect to increasing the penetration depth of dyes, antibodies and/or antibody mimetics, and/or labeled DNA and/or RNA strands into the thickness of a tissue specimen/section.